Development of an Integrated Point-of-Care Diagnostic Framework for Bloodborne Pathogens

Abstract

Diagnostic platforms are complex systems, integrating elements of assay chemistry, molecular biology, fluidics, and instrumentation for pathogen detection. Simplifying them is key to expanding their use to low-resource settings where infectious disease burden is the highest. In this thesis, I take a systems-approach to diagnostics development to leverage the strengths of innovations across technical domains into a platform for detection of bloodborne pathogens from a fingerstick sample. The first project demonstrates a highly multiplexed assay, designed to achieve coverage of HIV genetic sequence diversity by departing from empirical primer design strategies and employing a novel approach to multiplexing with cooperative neighboring assays. The second project developed an extraction-free simplification to complex sample preparation for bloodborne pathogens by directly diluting fingerstick plasma samples while scaling up reaction volumes to allow larger sample input and improved sensitivity. In the final project, I developed a prototype sample-to-result platform for detection of bloodborne pathogens from fingerstick samples. This integrated system was designed to leverage assay-level innovations in extraction-free amplification and the complementary strengths of centrifugal and paper-based microfluidics to create a platform for bloodborne pathogen detection.